摘要
目的:探讨miR-483-5p对人胰岛素样生长因子2(IGF2)基因P3启动子驱动的m RNA(P3 m RNA)表达的影响及其在肝细胞癌发生发展中的作用。方法:(1)采用real-time PCR检测人肝癌细胞株Huh7、Hep3B、Bel-7402、Hep G2和SMMC-7721,人正常肝细胞株HL-7702,83例人肝细胞癌组织和配对的癌旁组织,22例正常肝组织中miR-483-5p和P3 m RNA的表达水平,并应用Pearson相关分析评估P3 m RNA与miR-483-5p表达水平之间的关系。(2)将IGF2基因P3 m RNA的5’端非翻译区(5’UTR)克隆入p GL3启动子载体,构建P3 m RNA 5’UTR野生型(p GL3-P3-5’UTR-WT)及P3 m RNA 5’UTR突变型(p GL3-P3-5’UTR-MUT)重组萤光素酶报告质粒,将其分别与miR-483-5p mimic、miR-483-5p inhibitor及scrambled control共转染He La、293T及Huh7细胞,采用双萤光素酶报告系统检测萤光素酶活性。(3)分别将miR-483-5p mimic、miR-483-5p inhibitor及scrambled control转染Huh7及Hep3B肝癌细胞,应用real-time PCR检测这2种肝癌细胞P3 m RNA表达水平的变化。(4)应用real-time PCR检测肝癌细胞Huh7及Hep3B的细胞核和细胞质中miR-483-5p的表达水平;应用核连缀实验(nuclear run-on assay)分析miR-483-5p对P3 m RNA转录的影响;应用RNA稳定性实验分析miR-483-5p对P3 m RNA稳定性影响。(5)应用体外细胞功能实验研究miR-483-5p对Huh7肝癌细胞生长、凋亡、迁移与侵袭能力的影响。结果:(1)5种肝癌细胞株miR-483-5p及P3 m RNA表达水平均明显高于正常肝细胞株HL-7702(P<0.01),肝细胞癌组织中miR-483-5p及P3 m RNA表达水平均明显高于配对的癌旁组织及正常肝组织(P<0.01);线性相关分析显示,在5种肝癌细胞株及肝细胞癌组织中,P3 m RNA表达水平均与miR-483-5p水平呈正相关。(2)萤光素酶实验显示,miR-483-5p与P3m RNA 5’UTR的同源位点互补结合可促进P3 m RNA的表达。(3)瞬时转染实验显示,过表达miR-483-5p呈剂量依赖性促进Hep3B和Huh7肝癌细胞P3 m RNA表达水平的增高。(4)miR-483-5p表达实验显示,成熟miR-483-5p存在于肝癌细胞Hep3B和Huh7的细胞质和细胞核中;核连缀实验显示,miR-483-5p诱导Huh7肝癌细胞核中新生P3 m RNA转录;RNA稳定性实验表明,miR-483-5p不改变Huh7肝癌细胞P3 m RNA稳定性。(5)体外细胞功能实验显示,miR-483-5p促进Huh7肝癌细胞增殖,抑制其凋亡,并增强迁移与侵袭能力。结论:miR-483-5p高表达可部分通过上调IGF2基因P3 m RNA转录促进肝癌细胞生长、迁移与侵袭,进而参与肝细胞癌发生。
AIM:To investigate the effect of miR-483-5p on P3 promoter-driven mRNA(P3 mRNA)expression of human insulin-like growth factor 2(IGF2)gene and its role in the development of hepatocellular carcinoma(HCC).METHODS:The expression levels of miR-483-5p and P3 mRNA were analyzed by real-time PCR in human HCC cell lines Huh7,Hep3B,Bel-7402,HepG2 and SMMC-7721,normal human liver cell line HL-7702,83 cases of human HCC tissues and their matched adjacent nontumorous tissues(MANT),and 22 cases of normal adult liver tissues(NALT).The association between P3 mRNA level and miR-483-5p level was evaluated by Pearson correlation analysis.The full-length sequences of 5’UTR of P3 mRNA containing wild-type and mutant miR-483-5p-binding sequences were cloned into pGL3 promoter vector,which were termed pGL3-P3-5’UTR-WT and pGL3-P3-5’UTR-MUT,respectively.These luciferase reporter constructs were transfected into HeLa,293T and Huh7 cells together with miR-483-5p mimic,miR-483-5p inhibitor or scrambled control,and the luciferase activity was measured using dual-luciferase reporter system.The miR-483-5p mimic,miR-483 inhibitor and scrambled control were also transfected into Huh7 cells and Hep3B cells,and P3 mRNA level was detected by real-time PCR.The expression levels of miR-483-5p in the nuclear and cytoplasmic fractions of Hep3B cells and Huh7 cells were detected by real-time PCR.The effect of miR-483-5p on P3 mRNA transcription was evaluated by nuclear run-on assay.The effect of miR-483-5p on the stability of P3 mRNA was analyzed by RNA stability assay.Furthermore,the effects of miR-483-5p on the viability,apoptosis,migration and invasion of Huh7 cells were investigated.RESULTS:Significamtly high levels of miR-483-5p and P3 mRNA were detected in the 5 human HCC cell lines and the human HCC tissues as compared with the human normal liver cell line HL-7702,and the MANT and NALT,respectively.Linear correlation analysis revealed that P3 mRNA level was positively correlated to miR-483-5p level in the 5 human HCC cell lines and the human HCC tissues.miR-483-5p directly recognized the P3 mRNA 5’UTR to promote gene expression.Overexpression of miR-483-5p resulted in a significant increase in P3 mRNA expression in a dose-dependent manner in the Huh7 cells and Hep3B cells.The mature miR-483-5p was present in both cytoplasm and nucleus of Hep3B cells and Huh7 cells.miR-483-5p induced nascent P3 mRNA transcription in the nucleus of Huh7 cells.miR-483-5p did not alter P3 mRNA stability in Huh7 cells.Furthermore,miR-483-5p led to increased viability,apoptosis inhibition,and enhanced migration and invasion abilities in the Huh7 cells.CONCLUSION:High expression of miR-483-5p promotes the growth,migration and invasion of HCC cells in part through up-regulating P3 mRNA transcription,and is consequently involved in the development of HCC.
作者
马彦
朱翠萍
胡建军
李悅行
王敏
汤绍辉
MA Yan;ZHU Cui-ping;HU Jian-jun;LI Yue-xing;WANG Min;TANG Shao-hui(Department of Gastroenterology,The First Affiliated Hospital,Jinan University,Guangzhou 510632,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2018年第4期577-584,共8页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81572369)
广东省自然科学基金资助项目(No.2015A030313310)
中央高校基本科研业务费专项资金资助项目(No.21615462)